DESCRIPTION: Proper healing of corneal wounds is vital to maintaining a clear, healthy cornea and preserving vision. The long-term goal of this project is to understand the mechanisms involved in corneal epithelial wound repair. Recent data suggest that EGF receptor (EGFR) is present in the basal cell layer across the cornea, with a high concentration in the periphery and limbal cells. EGFR is activated within 15 minutes after making a corneal debridement. TGF-B receptors types I (RI) and II (RII) are localized in limbal basal cells of unwounded corneas. Following a corneal debridement, both RI and RII are dramatically upregulated and localize to the leading edge of the migrating epithelial sheet. These data suggest that the regulation of epithelial proliferation may be determined by the interplay between mitogenic signaling by the EGF family and antimitogenic signalling by the TGF-B family. The current application will test the hypothesis that diffusible growth factors regulate cell proliferation both during normal homeostasis and during wound healing by stimulating (via EGFR) and inhibiting (via TGF-BR)the proliferative cell cycle. Biochemical and molecular biological techniques will be used to test three specific aims. First EGFR, TGF-BR and their ligands will be localized and their expression quantified in wounded and unwounded corneas. The ratio of the levels of EGFR to TGF-BR in the limbal, peripheral, and migrating epithelia will be correlated with the rate of proliferation in these areas of the cornea. Second, the auto- and cross- regulation between the EGF and TGF-B receptors and their ligands will be determined. Third, the effect of EGF and TGF-B family members on the cell cycle will be determined by examining the effects of the growth factors on the expression and activity of the cell-cycle-associated proteins: retinoblastoma protein, cyclin-dependent kinase 2, cyclin-dependent kinase 4, cyclin D, and cyclin E. In addition, the effects of EGF and TGF-B on the expression of the cell cycle inhibitors, p15, p21, and p27 will be determined. Techniques to be used include western blotting, ribonuclease protection assay, RT-PCR, immunofluorescence microscopy, and in situ hybridization. The proposed studies will examine the positive and negative regulation of the proliferative cell cycle that occur during wound repair. These experiments will result in basic information required for developing therapeutics to be used to enhance impaired healing responses.